Thesis (D.Sc.D.)--Boston University, Goldman School of Dental Medicine, 2008 (Dept. of Periodontology and Oral Biology). / Includes bibliographical references: leaves 140-148. / Lysyl oxidases constitute a family of enzymes responsible for the formation of cross links in collagen and elastin. These enzymes have also been linked to pathological fibrosis. The importance of collagen in the structural and mechanical properties of bone led us to investigate the hypothesis that the absence of one or more of these enzymes could lead to a significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate it could facilitate the development of drugs that control its activity in pathological fibrosis. Bones from 12-week old mice (8 males and 8 females) with the compound genotype LOX+/-, LOXLl -/- were analyzed. 5 males of the genotype LOX+/+, LOXLl-/were also analyzed. 16 wild type mice (8 males and 8 females) were used as controls. μCT was used to analyze the trabecular and cortical bone morphology of both left femur and L5 vertebrae (n=5). The femora were subsequently subjected to mechanical testing using the twist failure in torsion. Right femurs (n=5) were used for histology and histromorphometric analysis. Tibia and fibula (n=5) were used for cross-link analysis. Two way factor ANOV A with post-hoc Tukey HSD test was used for statistical analysis. A P value of less than 0.05 was used to declare significance. μCT analysis of the trabecular bone in femur distal ... [TRUNCATED]

Dissertation (MSD) --Boston University, Goldman School of Dental Medicine, 2012 (Department of Oral Biology). / Includes bibliographic references: leaves 71-77. / Lysyl oxidases constitute a family of enzymes responsible for the formation of crosslinks in collagen and elastin. These enzymes have also been linked to pathological fibrosis. The importance of collagen in the structural and mechanical properties of bone led us to investigate the hypothesis that the absence of one or more of these enzymes could lead to a significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate it could facilitate the development of drugs that control its activity in pathological fibrosis. [TRUNCATED]

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Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 3 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5) Publico-12837c.pdf: 1773766 bytes, checksum: 56addbd5601cb4e7336afb640ed56fe8 (MD5) / Objetivo: O prolapso de órgãos pélvicos (POP) resulta da falha na sustentação do assoalho pélvico, e anormalidades do tecido conjuntivo podem estar envolvidas na etiologia e/ou na progressão da disfunção. Analisar-se-á a expressão diferencial de genes e de proteínas que participam da biossíntese do colágeno e da elastina: lisil oxidases (LOXs), fibulina-5, fibrilinas-1 e -2 e pró-colágeno C proteinase (PCP/BMP1), no tecido vaginal de mulheres sem e com POP acentuado consoante seu estado hormonal. Casuística e Métodos: Durante a histerectomia total, biópsias de parede vaginal anterior foram obtidas de mulheres caucasianas na pré-menopausa (fase proliferativa do ciclo menstrual) e na pós-menopausa com POP acentuado (POPQ estadio III e IV), e de controles assintomáticas (POPQ 0). RNAm e proteínas totais foram extraídos usando Trizol e RIPA Buffer, e os genes e proteínas de interesse quantificados por RT-PCR em tempo real e Imunobloting, respectivamente. As seguintes análises comparativas foram realizadas: (1) expressão dos genes e das proteínas da família LOX (LOX e LOXL1-4), fibulina-5 e fibrilinas- 1 e -2 em pacientes na pré-menopausa com e sem POP; (2) expressão do gene e da proteína PCP/BMP1 em pacientes na pré- e pós-menopausa com POP, e respectivos controles. Os testes de Wilcoxon signed-rank e Fisher foram usados para as análises estatísticas (p<0.05). Resultados: Obtivemos amostras de 15 pacientes e 11 controles na pré-menopausa para o estudo (1), e 39 pacientes na pré-menopausa (POP=23 e Controle=16) e 18 na pósmenopausa (POP=13 e Controle=5) para o estudo (2). A partir das análises, observamos (1) diminuição significativa na expressão dos genes LOX, LOXL1 e LOXL3, bem como nas proteínas LOX e LOXL3 no tecido vaginal de pacientes POP na pré-menopausa comparadas com mulheres assintomáticas (p<0.05); (2) hipoexpressão do gene PCP/BMP1 nos tecidos vaginais de mulheres com POP acentuado comparadas com controles, tanto na prémenopausa como na pós-menopausa (ambos p=0.01); redução significativa das isoformas 130kDa, 92,5kDa e 82,5kDa da PCP/BMP1 no tecido vaginal de pacientes na pósmenopausa (p=0.01), bem como hiperexpressão da isoforma 130kDa nas mulheres com POP acentuado na pré-menopausa (p=0.009), comparadas com as respectivas controles. Conclusão: As expressões das enzimas LOXs e pró-colágeno C proteinase estão alteradas no tecido vaginal de mulheres com POP, e são moduladas pelo estado hormonal. A alteração na regulação destas enzimas, envolvidas na biossíntese da matriz extracelular, pode contribuir para deficiente síntese do tecido conjuntivo e do suporte vaginal, e estar envolvida no desenvolvimento do POP. / Objective: Pelvic organ prolapse (POP) results from the failure of pelvic floor support, and connective tissue abnormalities may be involved in the etiology and/or progression of the dysfunction. We aimed to analyze the differential expression of genes and proteins related to the collagen and elastin biogenesis: lysyl oxidases (LOXs), fibulin-5, fibrillin -1 and -2, and procollagen C proteinase (PCP/BMP1) in vaginal tissue of women without and with advanced POP controlled by hormonal status. Materials and Methods: During total hysterectomy, anterior vaginal wall biopsies were obtained from Caucasian premenopausal women (proliferative phase of menstrual cycle) and postmenopausal women with severe POP (POPQ stage III and IV) and asymptomatic controls (POPQ 0). Total mRNA and protein were extracted using Trizol and RIPA buffer, and the genes and proteins of interest were quantified by real-time RT-PCR and Immunoblotting, respectively. The following analysis were performed: (1) expression of LOX family genes and proteins (LOX and LOXL1-4), fibulin-5, fibrillin-1 and -2 in premenopausal women with and without POP; (2) PCP/BMP1 gene and protein expression in vaginal tissue of pre- and postmenopausal POP women, and respective controls. Wilcoxon signed-rank and Fisher tests were used for statistical analysis (p<0.05). Results: Samples from 15 premenopausal patients and 11 controls were obtained for study (1); 39 premenopausal (POP=23 and Control=16) and 18 postmenopausal women samples (POP=13 and Control=5) for study (2). We observed: (1) significant decrease in expression of LOX, LOXL1 and LOXL3 genes, as well as LOX and LOXL3 proteins in vaginal tissue of premenopausal POP patients compared with asymptomatic women (p<0.05); (2) PCP/BMP1 gene downregulation in the vagina of women with severe POP compared with controls, in both premenopausal and postmenopausal phase (both p=0.01); significant reduction of 130 kDa, 92.5 kDa and 82.5 kDa PCP/BMP1 isoforms in vaginal tissue of postmenopausal patients (p=0.01), and 130 kDa isoform upregulation in premenopausal women with severe POP (p=0.009), compared with their respective controls. Conclusion: The expression of LOXs enzymes and PCP/BMP1 are altered in vaginal tissue of women with severe POP, and are modulated by hormonal status. Dysregulation of these enzymes involved in the extracellular matrix biogenesis may contribute to impaired tissue and vaginal support, and may be involved in POP development. / TEDE / BV UNIFESP: Teses e dissertações